Rocha (1) argues that the experimental approaches we used (2) to conclude that intestinal intraepithelial T lymphocytes (IELs) expressing TCRαβ are largely derived from thymus, rather than from cryptopatch cells, leave room for alternative explanations. We made our argument based on two separate sets of results. First, we showed that lin–c-kit+IL-7Rα+ cryptopatch cells, which express the transcription factor RORγt, failed to develop in its absence, but IELs, particularly those expressing TCRγδ, were nevertheless present. Based on this finding, we were unable to rule out that other local progenitors, distinct from cryptopatch cells, gave rise to IELs. However, our results clearly showed that cryptopatch cells were not involved in this process. Our gating for lineage markers was identical to that described by Saito et al. (3) in their original description of a cryptopatch origin for IELs. Moreover, we did not observe any RORγt+c-kit+IL-7Rα+ cells in the Lin+ gate. The “local T cell–committed precursors” described by Rocha may indeed have been spared in the absence of RORγt, but we do not believe that such cells form the clusters that characterize cryptopatches in the lamina propria. In the absence of RORγt, there was no visible restoration of cryptopatch structures by expression of a Bcl-xL-IRES-YFP transgene under Rorc(γt) control, despite restoration of normal numbers of TCRαβ IEL (2). If these IELs were derived locally from c-kit+IL-7Rα+ precursors, it would then be necessary to invoke expansion of such RORγt– precursors by a cell nonautonomous effect of Bcl-xL, because we were unable to observe GFP (green fluorescent protein) /YFP+ (yellow fluorescent protein) cryptopatch cells. By immunohistochemical analysis, we have found that all c-kit+ cells within cryptopatches express RORγt. These observations leave open the formal possibility that RORγt-negative cells that reside outside cryptopatch structures and have the c-kit+IL-7Rα+B200+ phenotype described by Rocha (1) may give rise to IELs.

In the second set of experiments, we used genetic fate mapping to show that TCRαβ IELs are derived from thymic rather than from intestinal cryptopatch precursors. We bred mice in which Cre recombinase was under the control of murine CD4 transcriptional elements with reporter mice in which GFP is expressed only after Cre-mediated excision of a transcriptional STOP sequence. In progeny, TCRαβ IELs expressed GFP, as did cells in the thymus, but there was no expression in intestinal lin–c-kit+IL-7Rα+ cells, which suggests that such cells are unlikely to be progenitors of these IELs. Rocha correctly points out that there can be a temporal dissociation between Cre-mediated deletion either of coding sequences (as in the case of conditional knockouts) or of a transcriptional STOP sequence (as in the case of fate mapping) and the respective loss or gain of expression of the relevant gene product. She cites as an example the commonly used CD4-Cre mouse, in which deletion is first observed late in CD4–8–(DN) thymocytes, after β-selection (4, 5). Depending on multiple factors, such as the half-life of the relevant gene product, the defect may be manifested only later, in double-positive (DP) thymocytes. Rocha argues that, in CD4-Cre mice, the recombinase may be activated in T cell precursors in the gut or elsewhere outside of thymus. This is theoretically possible, and we cannot absolutely rule it out. However, we feel that it is highly unlikely that Cre activation occurred in intestinal lin–c-kit+IL-7Rα+ cells or in mature IELs. In CD4-Cre mice crossed to R26R mice, we observed GFP expression in DN4 stage cells, which suggests that GFP is expressed soon after excision of the transcriptional STOP sequence (Fig. 1). In addition, the Cre transgene expressed under control of the Rorc(γt) regulatory elements induced expression of the GFP reporter in intestinal lin–c-kit+IL-7Rα+ cells (2). Taken together, these observations make it rather unlikely that Cre is expressed in lin–c-kit+IL-7Rα+ cells under control of the CD4 expression cassette but activates the GFP reporter only at a later time. Rocha also suggests that our observation could be due to expression of Cre in the TCRαβ IELs. In many transgenic strains made with this expression cassette, we never observed expression outside DP thymocytes and CD4 lineage T cells, and we are not aware that others have observed ectopic expression. The GFP marking of TCRαβ but not TCRγδ cells in the intestine is thus most consistent with the former population developing from thymic DP precursors and not from intestinal cryptopatch cells. We agree, however, that ectopic expression of Cre in a small population of intestinal intermediate precursors for TCRαβ (but not TCRγδ) IELs cannot be formally ruled out. If such intermediate precursors exist in normopenic mice, then they would be present only outside organized cryptopatches, based on the first set of results discussed above.

Expression and activity of the CD4-cre transgene in thymocytes. EGFP (enhanced GFP) expression in gated thymocyte subpopulations from ROSA26R (filled histograms) and ROSA26R;CD4-cre (open histograms) mice shown. Thymocytes were stained with antibodies to CD4, CD8, CD25, and CD44. Gates for CD4+CD8+ DP thymocytes and CD4–CD8– subpopulations are shown in the left panels. The CD4–CD8–CD44+CD25– population contains fractions of mature TCRαβhi cells and NKT cells that are derived from CD4+CD8+ DP thymocytes (8).

Our findings did not exclude an extrathymic origin for some IELs, in particular, TCRγδ T cells. Although intestinal precursors for T cells have been described (6, 7), their contribution to the generation of the intestinal T cell pool in normal mice remains poorly assessed. Our results suggest that this contribution is minimal in healthy mice; however, it could be substantial in lymphopenic mice or in the setting of intestinal inflammation in which large numbers of T cells are mobilized. More experiments are required to determine whether, under such circumstances, cryptopatch cells function as T cell precursors. We believe, however, that the principal function of the cryptopatch cells is to induce formation of lymphoid follicles in the intestinal lamina propria in a manner similar to induction of lymph nodes and Peyer's patches by the RORγt-expressing fetal lymphoid tissue inducer cells.